Gene expression occupies a key function in the evaluation of molecular processes in the body and great efforts are being made to investigate the significance of the expression of numerous genes also as a result of drug effects and to check it with respect to its predictive value in regard to the course of an illness and the success of a therapy.
A prerequisite for converting genetic information initially is the transcribing of the corresponding DNA sequence into mRNA. Gene expression can be regulated at the level of transcription as well as post-transcriptionally. For evidence-based medicine, the clarification of the mechanisms, which lead to a changed gene expression in the course of an illness, is an important objective, because new therapy concepts can be derived from it, which lead to an improved treatment of patients.
According to estimates of the organizers, the human genome project presumably will come to a conclusion in the year 2001. At the end of this project, which is being conducted worldwide, approximately 140,000 genes will have been identified. The analysis of the gene expression in different cell types and tissues, which provides important information concerning the normal state and the genesis of the pathologic state of cells and tissues, represents the greatest challenge at the present time as well as in the post-genome epoch.
A plurality of methods is employed for the analysis of gene expression, including Northern Blot and RT-PCR techniques. By means of chip-based technologies, that is, planar carriers of plastic, glass, gelatin, etc., on the surface of which a plurality of different (DNA) molecules, the positions of which are known and can be addressed, are disposed, thousands of genes can be investigated simultaneously with respect to their expression.
Aside from the different methods of analyzing gene expression, new technologies are being developed, in order to be able to detect nucleotide polymorphisms (that is, different variations of a gene) systematically, and to be able to evaluate them with respect to their biological significance in the course of an illness and, in the case of a medical application, in the sense of an individualized therapy.
A relatively recent method for fluorescence-based gene expression analyses and gene mutation analyses is represented by the investigation of amplified probes by the PCR technique in real-time PCR analytical equipment, such as the Lightcycler (Roche Diagnostics), TaqMan (Perkin-Elmer), etc.
The LightCycler is equipped with a three-channel fluorimeter, which can detect fluorescence at 530 nm (SYBR-Green), 640 nm (LC-RED 640) and 705 nm (LC-RED 7050). The manufacturer, Roche Diagnostics makes several tests available for the PCR amplification in the LightCycler, which can be used depending on the type of labeling (SYBR-Green or FRET methods; see below).
Dye Labeling
In order to be able to measure the newly synthesized in DNA in the subsequent real-time PCR, the DNA must be labeled by suitable dyes, which can be detected. For detecting the fluorescence signals, various real-time PCR detection systems were developed, with which it is possible to follow the whole of the PCR reaction. In the following, basic principles of the detection are explained:
1.) Fluorescence resonance energy transfer (FRET) is a process, for which a donor molecule, fluorescing after being stimulated by short-wave light, transfers its emission energy to a second acceptor molecule, which reacts to this with the emission of light of longer wavelength. The energy transfer from the one to the other molecule takes place over electron flow. The reporter molecule provides information concerning the product increase during the PCR. The so-called quencher molecule absorbs the fluorescence signals of the reporter molecule as long as both molecules are directly adjacent to one another in the hybridization probe. In this basic state, the reporter emission radiation for the fluorescence detector, with which the product increase in the PCR is measured, is invisible. Only as the PCR product increases, is there a spatial separation from the reporter and the quencher molecule. By these means, the reporter fluorescence becomes detectable and correlates directly with the amount of PCR product formed in the reaction.
A further method is referred to as the TaqMan method. In the case of the TaqMan method (or 5xe2x80x2-nuclease assay), the fluorescence-labeled hybridization probes bond to the complementary target strand between the primer binding sites. For the synthesis of the new strand, the hybridization probe is cut into small fragments by the 5xe2x80x2-3xe2x80x2-exonuclease activity of the Taq polymerases and released from the target strand. The reporter molecules and the quencher molecules are now present separately in the reaction mixture and the measured increase in the reporter fluorescence per PCR cycle correlates directly with the increase in the PCR product.
Other hybridization probes, synthesized according to the FRET principle, can be used for carrying out mutation analyses. Particularly important is the detection of so-called single nucleotide polymorphisms (SNPs), which come about due to the exchange of individual bases. Two hybridization probes, each labeled with a fluorescence dye, are used for the SNP analysis.
One donor probe binds directly adjacent to the mutation region. A second probe is produced so that it binds either complimentarily to the wild type or over the mutation site. In the melting point analysis, carried out after the PCR, the probe melts off at a particular temperature. If the probe bonds complimentarily to the wild type, it melts off at higher temperatures. On the other hand, in the presence of a mutation, the probe melts at lower temperatures. A mutation analysis therefore becomes possible. The fluorescence decline is calculated as a negative first derivative (as a melting peak). The mutation can be diagnosed by the displaced melting curve. This method with different fluorescence dyes can only be used by means of a real time detection system.
The principle of the real time detection system also forms the basis of the LightCycler from Roche Diagnostics. The LightCycler has three channels by means of which the emitted light quanta of dyes can be detected. DNA can by labeled with SYBR-Green; in addition, FRET probes, which are labeled with LC-Red 640 or LC-Red 705 dyes can also be used.
If the FRET method is used in the LightCycler, special hybridization probes must be added to the reaction mixture. They are labeled with fluorescein and LC red 640 or LC red 705 (from Roche Diagnostics). The fluorescence is observed only if both probes (donor probe and acceptor probe) have bonded in the immediate spatial vicinity to the target sequence. The transfer of light quanta (h*v), namely the fluorescence resonance energy transfer (FRET; see FIG. 1), then comes about.
2.) In the SYBR-Green method, the SYBR-Green intercalates in each case between two complementary base strands during the DNA synthesis and, with that, experiences a measurable increase in fluorescence as the PCR reaction progresses (see FIG. 2). However, the use of SYBR-Green lacks any specificity with regard to the template, which is to be investigated (that is, the DNA binding site), because the primer dimers, which are formed during the reaction, also cause an increase in fluorescence. Initially, this cannot be differentiated from the desired DNA synthesis product and can lead to wrong interpretations. However, it is possible to differentiate between the specific product and primer dimers at the end of the PCR by means of a melting curve analysis. For this, the PCR products are heated continuously over a particular temperature range and are present only as a single strand, depending on their melting point. The decrease in fluorescence, associated with this, is recorded. Smaller fragments, such as the primer dimers, have a melting point, which is lower than that of larger PCR products.
The representation of the fluorescence signal changes as a function of the temperature, derived from this, results in a curve, in which the specific PCR product becomes distinguishable from the primer dimers, if the melting points differ clearly from one another.
PCR Amplification in General
With the help of the polymerase chain reaction (PCR), clearly defined DNA sections of a gene can be reproduced million fold. For this purpose, two oligonucleotides (primers), which are complementary to the target sequence and each of which adds to one of the DNA strands, are added to the PCR reaction mixture. Moreover, sufficient amounts of the four desoxynucleoside triphosphates, a certain amount of magnesium chloride and a heat-stable DNA polymerase are added to the reaction cocktail. The individual substances for the PCR reaction are offered by numerous companies (Roche Diagnostics, Qiagen, Promega, Stratagene, TaKaRa, etc.). Previously prepared reaction mixtures or xe2x80x9cMasterMixesxe2x80x9d are also offered, to which only the primers and the DNA, which is to be investigated, have to be pipetted.
However, because of the lower sensitivity and selectivity and due to the formation of primer dimers, which are false DNA synthesis products, the informative power of the fluorescence-based gene expression analyses and gene mutation analyses, using our own PCR reaction mixtures with the conventional components and concentrations or using the commercially obtainable MasterMixes, is very limited. The formation of primer dimers must be emphasized especially, since it can lead to false findings, as a result of which appreciable risks arise for the patient and for biomedical research in general and therefore a reliable medicinal diagnosis, for example, during accompanying investigations in the course of the therapy, cannot be guaranteed. Furthermore, these investigations are very cost intensive, especially when the Roche kits are used, as a result of which the number of possible investigations is greatly limited by the respective research budget.
It is therefore an object of the invention to increase the selectivity and sensitivity of fluorescence-based gene expression analyses and gene mutation analyses and, by suppressing the formation of primer dimers (see FIG. 3), to prevent wrong diagnoses and erroneous findings.
An increase in the selectivity, sensitivity and the suppression of primer dimer formations, fluorescence-based gene expression analyses and gene mutation analyses is accomplished pursuant to the invention owing to the fact that bovine serum albumin is added to the conventional PCR reaction components and that the magnesium chloride concentration is adjusted accurately depending on the Taq polymerase used. In SYBR-Green DNA labeling, the exact adjustment of the concentration is indispensable for realizing the inventive responses (sensitivity, selectivity).
The use of the inventive PCR reaction mixture leads to a clearly improved sensitivity and selectivity of fluorescence-based gene expression analyses and gene mutation analyses in the animal, bacterial, vegetable and human genome and prevents wrong diagnoses. By these means, it is possible to carry out such detections or investigations on samples, which previously could not be analyzed in this way because of their low RNA or DNA concentration. Moreover, the claimed invention leads to a dramatic reduction in the costs of the investigation.
The inventive PCR reaction mixture makes possible a distinct increase in the information power of the semiquantitative and totally quantitative determination of the gene expression in tissues and organs in the healthy, diseased and medicinally affected state. Moreover, because of the possibility of using inexpensive components in gene expression analyses or the detection of nucleotide polymorphisms, the use of the inventive PCR reaction mixture leads to a reduction in costs from about DM 4.13 per sample to DM 0.75 per sample.
The technical area of application of the invention comprises, above all, a) the pharmacogenomics and here, especially the discovery of genomic targets for drug candidates in research and development, or for products already introduced on the market, b) the detection of nucleotide polymorphisms, especially in the molecular diagnosis of diseases based on gene mutation analyses and gene polymorphism, in drug therapy and here, in particular, in the individualized dosing of drugs and for the rational interpretation of the pharmacokinetic course of a therapy, c) for the characterization of potential drugs at the gene expression level, d) the toxicogenomics and here, especially, the use in the case of toxicological investigations for preclinical development and for predicting toxic effects and for the toxicological characterization of individual materials and material mixtures at the gene expression level, e) the molecular diagnosis and here, especially the screening and the diagnosis of genes relevant to the illness, the monitoring of the course of an illness and a therapy and the molecular prognosis of diseases and f) the research and here, in particular, the identification of molecular interactions of materials, material mixtures and biological agents on the genome level, the identification of gene intercalations and the function analysis of new genes, including sequence analyses and gene clonings.